Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. These cited publications are incorporated by reference herein, in its entirety and for all that they illustrate.
Selective chemical control of biochemical processes within a living cell enables the study and modification of natural biological systems in ways that may not be obtained through in vitro experiments (Cook et al., C.R. Bioorg. Med. Chem. 10: 829, 2002; Chen et al., Curr Opin Biotechnol. 16:35, 2005). Accordingly, access to promiscuous metabolic pathways has provided a unique chemical entry into small molecule engineering in vivo (Mahal et al, C.R. Science 276:1125, 1997). A method for covalent reporter labeling of carrier proteins using promiscuous phosphopantetheinyltransferase (PPTase) enzymes and reporter-labeled coenzyme A (CoA) has recently been described (La Clair et al., M.D. Chem Biol. 11:195, 2004). Until now, this method has been limited to in vitro and cell-surface protein labeling, as CoA derivatives have not been shown to penetrate the cell (La Clair et al., M.D. Chem Biol. 11:195, 2004; Mercer et al., Chem Bio Chem 2005; George et al., Am. Chem Soc., 126:8896, 2004; Yin et al., Am Chem Soc. 126:13570, 2004; Yin et al., Am Chem Soc. 126:7754, 2004). To overcome this obstacle, labeled metabolic precursors may be delivered to the cell culture, which results in cellular uptake and metabolic conversion into active, labeled CoA derivatives. In the process, a chemoenzymatic route to protein modification via a four-step sequence is established.
The chemical synthesis and activity of CoA has been studied for well over a century, yet the full biosynthesis of the cofactor has only recently been elucidated in prokaryotes and eukaryotes (Mishra et al., Chem Rev., 100:3283, 2000, Mishra et al., Bacteriol., 183:2774, 2001; Martin et al., Biochem. Biophys. Res. Commun. 192:1155, 1993; Strauss et al., Biol. Chem. 276:13513, 2001; Daugherty, et al., Biol. Chem. 277:21431, 2002; Kupke et al., Biol. Chem. 278:38229, 2003; Zhyvoloup et al., Biol. Chem 277:22107, 2002 and Worrall et al., Biochem. 215:153, 1983). CoA is biosynthesized from vitamin B5 by five enzymes in E. coli, CoaA-CoaE, while eukaryotes contain a fusion of CoaD and CoaE, PPAT/DPCK. Knowledge of the substrate specificity of these enzymes remains incomplete, although some evidence points to promiscuity within this pathway. Early studies on CoaA indicated that the enzyme will also accept pantetheine as a substrate (Abiko, Biochem 61:290, 1967 and Shimizu et al., Biol. Chem 37:2863, 1973). This ability has since been used in the chemoenzymatic synthesis of CoA analogs (Rudik et al., Biochemistry 39:92, 2000; Martin et al., J. Am. Chem. Soc., 116:4660, 1994; Schwartz et al., Biochemistry 34:15459, 1995 and Nazi et al., Anal. Biochem., 324:100, 2004). This permissiveness suggests the ability of the CoA biosynthetic pathway to convert reporter-labeled pantetheine to reporter-labeled CoA in vivo. To this end, the synthesis of fluorescently-labeled pantetheine analogs provides a direct link to reporter-labeled post-translational modifications (Mandel, A L et al. 2004).